Arthritis is a painful and often crippling disease that initially results in painful, swollen, and inflamed joints. It often progresses to deform or completely destroy joints that then require replacement. This disease is a result of the body mistakenly attacking type II collagen, which is the major structural component of cartilage tissue. One function of cartilage tissue is that it serves as a lubricant in the joints, keeping bone from rubbing on bone. As the disease progresses and more of the cartilage is destroyed, bone does begin to wear on bone. The two most prominent types of arthritis are rheumatoid arthritis and osteoarthritis. The usefulness of undenatured type II collagen has been shown in ameliorating the symptoms of osteoarthritis in humans (Int. J. Med. Sci., 6(6), 2009, 312-321), horses (J. Vet. Pharmacol. Ther., 32, 2009, 577-584), and dogs (J. Vet. Pharmacol. Ther., 28, 2006, 385-390), which are all here fully incorporated by reference.
In order to initiate rheumatoid arthritis, it appears that an individual must have an inherent susceptibility. There is now evidence that, in susceptible people, this disease is initiated by exposure to the relatively common Epstein-Barr virus. The ability of the Epstein-Barr virus to initiate rheumatoid arthritis has been linked to a key amino acid sequence which is identical to a sequence found in human type II collagen. Thus, in generating antibodies to destroy the Epstein-Barr virus, the body generates antibodies that are also capable of attacking its own collagen.
Osteoarthritis has recently been found to also be an attack by the autoimmune system on cartilage. It is interesting that osteoarthritis occurs in animal species that do not, as a species, have rheumatoid arthritis. These species include canine, such as dogs, and equine, such as horses. Osteoarthritis is strongly related to age in both animals and humans. One likely reason for this age-related effect is an alternate method for the autoimmune system to be activated to initiate an attack on the body's cartilage. Such activation method may involve the very life-sustaining act of metabolism. In order to convert carbon-based food into CO2 and energy, the body creates massive numbers (many millions) of reactive oxygen species (ROS). In this process of breaking down food and producing energy, DNA chain breakage or other damage is said to occur over a million times per day in the average person. A large portion of this damage relates to cleavage of DNA, which is quickly repaired or destroyed by the immune system. This very routine action of the body's immune system is absolutely vital to human life. Some of the damaged molecules have the potential to cause the immune system to generate antibodies that are then capable of attacking the body's own collagen. When a body ages, a combination of effects causes it to become more susceptible to this osteoarthritis inducement. In some cases the DNA damage repair system becomes weaker. In other cases there may simply be a gradual build up and accumulation of the water-soluble molecular fragments that have the potential to generate antibodies that are capable of attacking the body's own collagen. When a sufficient accumulation of these water-soluble molecular fragments occurs, the collagen-destroying antibodies are activated.
In order to study the effect of proposed techniques for amelioration of arthritis, it is necessary to have arthritic animals. Two techniques to artificially induce arthritis in rats have been developed. These inducements have been accomplished more quickly than for osteoarthritis in humans, but in a similar manner. With rats the newer technique is by intradermal (under the skin) injection of a broken-down, water-soluble fragment of undenatured type II collagen (extracted from chicken cartilage). This technique has been termed collagen-induced arthritis (CIA). The second and older technique is accomplished by intradermal injection of the well-known Microbacterium tuberculosis (MT).
It has also been shown that rats can be prevented from getting arthritis via inducement or the effects of inducement can be greatly reduced. This prevention is accomplished by ingestion (or arterial injection) of the same broken-down, water-soluble, difficult-to-prepare fragment of type II collagen for several days prior to the attempted inducement. Once arthritis has been induced, the effects of the disease can be reduced by the continual oral administration of the same broken-down, water-soluble fragment of type II collagen. In later clinical studies with humans having arthritis, oral administration of the broken-down, water-soluble fragment of type II collagen was is shown to be similarly beneficial in reducing the effects of the disease.
Oral administration of this broken-down, water-soluble, undenatured fragment of type II collagen represents the very first technique for amelioration of the symptoms of arthritis offering a reversal rather than simply a slowing of the progress of the disease. This oral technique is believed to effectively reverse the debilitating effects of arthritis by causing desensitization to type II collagen. After this desensitization the body slows or stops the production of antibodies that destroy its own collagen. This process has been called “oral tolerization”, which is a partially understood process that the body uses to stop a person's immune system from treating food as a hostile foreign body. If foreign proteins are introduced via the digestive system, the body automatically suppresses the immune system responses to these proteins. It is a technique that has been used in the past to ameliorate simple allergies, such as an allergic reaction to poison ivy or pollen.
Although this oral administration of broken-down, water-soluble, undenatured fragments of type II collagen represents a long-sought and highly desired technique for amelioration of the symptoms of arthritis, the broken-down, water-soluble fragments of type II collagen are difficult to prepare. Typically the fragments are extracted from the tiny sternal cartilages of 2.5-week-old chicks. In a preparation of this prior art, eighty animals were required to produce 19 g of cleaned sterile cartilage dissected free of surrounding tissue. It is typical of the prior art to perform up to seven operations, consisting of extractions or digestions, on each batch of tissue in order to obtain the broken-down, water-soluble fragment of type II collagen. The procedure of this prior art is thus seen to have several serious deficiencies. An extremely large number of animals is required to obtain a small amount of the desired product. The purification procedure is very time-consuming, requiring multiple extractions, digestions, and precipitations. Sometimes ultrafiltration of the final product is required as a final step to remove pathogens from the water-soluble product.
It was later discovered by Dr. Eugene Moore that it is not necessary to break the undenatured type II collagen into a water-soluble state to obtain the full antiarthritic effect when ingested. In U.S. Pat. Nos. 5,645,581; 5,637,321; 5,529,786; and 5,750,144 (which are hereby fully incorporated by reference) Moore surprisingly found that the normal digestive process is sufficient. That is, when the whole, undenatured cartilage is digested, the effective amino acid sequence is separated and passed into the blood stream where it can reduce the symptoms of arthritis. This accomplishes the same goal as the earlier experiments with rats where the desired effect was is obtained by direct injections of the water-soluble fragment into the blood stream. This 26 amino acid sequence has been identified and is presented by Trentham in U.S. Pat. No. 5,399,347 (which is hereby incorporated in full by reference). It was is shown that this sequence, though difficult to prepare from sequencing monomeric amino acids, has amelioration effects for arthritis.
In the above-referenced Moore patents it was is found preferable to utilize the much larger cartilage from young 4- to 6- or more month-old chickens. Such usage made makes more cartilage available that is also easier to maintain in a sanitary state. Although chicken cartilage is preferred, the above-referenced Moore patents teach that cartilage from other animal tissue containing type II collagen can be effectively utilized. Bovine or porcine cartilage, or vitreous humor of eyes, for example, can be used if desired, although solid cartilage is preferred and chicken sternal cartilage is most preferred. Moore's technique for preparation of cartilage for oral administration to humans consists of first dissecting the cartilage free of most or all surrounding tissues so that the cartilage can be, for example, diced into smaller pieces. The diced cartilage is then sterilized by means known in the art and, for example, formed into capsules containing therapeutic levels of type II collagen, said levels being at least about 0.01 gram and preferably from about 0.1 to about 0.5 gram of cartilage to provide a therapeutic dose. The use of more mature chickens in the Moore approach is surprising in view of the prior art which teaches only the use of chicks of less than 3 weeks of age. The usefulness of the more mature chickens allows an almost 100-fold increase in the amount of harvestable cartilage from a single animal. This, of course, makes the desired product more readily available in therapeutic quantities, and also greatly decreases the possibility of micro-contamination due to the reduced handling during separation from relatively fewer animals.
It is difficult to preserve cartilage in its native undenatured state and thus maintain its effectiveness in alleviating the symptoms of autoimmune diseases. In the past, it has been possible to preserve the cartilage by two techniques. For the first technique, the above-referenced Moore patents teach that the collagen can be preserved successfully for an extended period by cleansing, cooling, and storing the cartilage at very low temperatures. Cartilage stored in this manner does not experience the growth of harmful pathogens or change in structure of the collagen which would cause it to become denatured and thus lose its effectiveness. The cleansing state of this process is by sterilization using, for example, chlorine-producing agents and/or hydrogen peroxide. The second technique, shown in the work of Schilling et al. (U.S. Pat. No. 7,083,820 (which is hereby fully incorporated herein by reference) involves drying cleansed cartilage under special low-temperature conditions in the presence of an inorganic salt, which greatly extends the storage life of the collagen Schilling emphasizes the need for stable long-term storage for this cartilage in order to make its beneficial effects available to a wide range of the world's population. He points out the failing of all other known stabilization and storage methods in the following quotation from his patent. “Thus irradiation, thermal processing, the use of preservatives, freeze drying and other known methods of sterilization, are either ineffective or difficult to use to stabilize a product for human consumption which retains the original biologic structure of naturally occurring materials. Accordingly, there is an acute need for a novel method to retain the biologically active component in natural materials in its original form that can be delivered in a safe and cost-effective manner for human consumption and use by other life forms.” Schilling further points out the need for maintaining the biological stability for greater than 3 years in the following quotation. “Another major feature of this invention is that the resulting product maintains its microbial safety for greater than 3 years at room temperature storage conditions.”
During cleaning and preparation for use, the cartilage is difficult to completely free from biological contamination such as pathogens and other microbes, which is required to maintain its safety. These pathogens and other microbes must be removed to render the undenatured type II collagen fit for human or animal consumption, even after extensive storage. It is desired to have the cartilage free of additives and easy to handle, store, ship, and consume.
The use of carbon dioxide (CO2) gas to inhibit the growth of microorganisms and extend the storage life of fruits, vegetables, meats, and other short-term consumable food items is well known. This knowledge goes back to the time of the Romans who would pack caves with fruit to let the off-gasses due to ripening, largely CO2, accumulate and slow the ripening and thus prolong the eatable quality of the food. This extension in usefulness of the produce is sometimes measured in hours in the case of cut fruit, for example. Such extension in useful life is often measured in days for items such as meat and poultry. The inhibition in ripening for uncut apples and other thickly skinned whole fruits is often measured in weeks as has been shown, for example, by Daniels (J. Food Prot., 48 (6), 1985, 537-537, which is hereby fully incorporated by reference). Daniels also summarizes the use of CO2 as a means to merely retard the deterioration and spoilage of butchered meat or otherwise comminuted food types and thus increase, in a small way, the useful storage life.
Brecht (Food Tech., 34, 1980, 45-50), in another summary of the use of controlled atmosphere to retard spoilage of produce, cites some negative results on the use of gas phase CO2. Brecht reports acetaldehyde accumulation and ultrastructure alterations as examples that suggest that gas phase CO2 induces an uncontrolled breakdown of tissue. The product of the present invention surprisingly does not have these negative results.
Ogilvy (Food Tech., 5, 1951, 97-102) examines the effects of CO2 on prolonging the storage life of cut-up chicken. He uses the concentration of slime-forming bacteria reaching a count of 2×108 per square centimeter as an end of useful life. Ogilvy also notes a common, serious problem when CO2 is used to store meat or fish: discoloration, with an undesired dark brown color developing in poultry flesh. The product of the present invention is surprisingly void of such discoloration at even the highest CO2 levels. The product of the present invention is very white or clear unless purposefully colored with an added dye or other coloring material. Ansuetto et al. (paper presented at Institute of Food Technologists Annual Meeting, Anaheim Calif.; Jun. 10-13, 1984) examines strawberries, along with other produce. He cites data showing that strawberries are particularly susceptible to decay. He extends storage from less than 3 days to about 6 days using a 30% CO2 atmosphere in the packages. He also discusses some cases where higher levels of CO2 are harmful, where berries must be shipped with “scrubbers” such as lime to absorb the otherwise harmful levels of CO2.